| Objective:To observe the expression of lipopolysaccharide(LPS) induced renal tubular epithelial cells(HK-2) on the expression of mi R-494,and further explore the mi R-494 on LPS induced apoptosis in HK-2 cells regulation function and its mechanism. Methods:1. HK-2 cells were cultured in vitro,treat HK-2 with 1 ug/ml LPS for different time(0 h、1 h、3 h、6 h、12 h).Real-time PCR were used to assess the expression of mi R-494 after LPS treatment for different time.2. mi R-494 lentivirus vectors and negative control lentivirus vectors were constructed for transfecting HK-2 cells.The transfection efficiency was estimated by fluorescence microscope.All of the cultured HK-2 cells were divided into four groups: normal control group、LPS stimulation group、LV-Zs Green-mi R-494 +LPS group、LV-Zs Green +LPS group.In addition to the control group with no treatment,the rest of groups to be treated with 1ug/ml LPS for 6 h after giving each grop treatments respectively. Detected the apoptosis rate by Annexin V-FITC/PI detection kit.Real-time PCR were used to assess the expression of mi R-494 expression level.At the same time,the production of IL-6 and TNF-α protein in the supernatant of cells were assayed by enzyme-linked immunosorbent assay(ELISA). Results:1. The expression level of mi R-494 peaked at 1 h after LPS stimulation(3.19±0.21,P<0.05),the expression of 3 h、6 h and 12 h quantity gradually decline,all of group mi R-494 expression quantity has statistical significance than in 0 h group.2. HK-2 cells were transfected with the LV-mi R-494 lentivirus vectors successfully.Green fluorescent protein were using inverted fluorescence microscope after 72 h,Results showed that transfection efficiency were above 80%,and q PCR detection the expression levels of mi R-494 in LV-mi R-494 tranfected cells was 8.57±0.50( P < 0.05), the differences were statistically significant.The result Prompted transfection success.3. Compared with normal control group,the expression levels of mi R-494 in LPS stimulation group,LV-mi R-494+LPS group and LV+LPS group were(8.57±0.50 vs 2.47±0.04 vs 2.34±0.02),the differences were statistically significant(P<0.05). The expression levels of ATF3 in LPS stimulation group,LV-mi R-494+LPS group and LV+LPS group were(2.12±0.37 vs 5.84±0.42 vs 5.45±0.53),the differences were statistically significant(P<0.05).4. Flow cytometry to detect each group HK-2 level of apoptosis:Compared with normal control group, the oher three groups apoptosis rate were gradually increased.LV+LPS group and LPS group was no significant different(P>0.05).5. ELISA to detect the expression of IL-6 and TNF-α in HK-2 cells:After lipopolysaccharide treatment,the levels of IL-6 and TNF-α were obviously higher than normal control,and the LV-mi R-494+LPS group was obviously higher than the other two groups.,the differences were statistically significant(P<0.05),revealed the overexpression of mi R-494 increased LPS induced IL-6 and TNF-α secretion. Conclusion:1. HK-2 cells treated with LPS show a higher apoptosis level compared with control group.2. HK-2 cells were transfected with the LV-mi R-494 lentivirus vectors successfully,the expression of mi R-494 was significantly increased while its target gene ATF3 m RNA decreased.3. mi R-494 could increased the secretion of inflammatory cytokines from HK-2,and promote from LPS-induced HK-2 cells apoptosis,the mechanism may due to inhibition its target gene ATF3. |
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